The magnitude of the dephosphorylation of glucose-6-phosphate in brain in vivo was reexamined in this report. Qualitatively, the reaction was examined by comparing the ratio of 3H-glucose/14C-glucose in brain and blood from 1 to 10 min after an injection of [2-3H]- and [U-14C]-glucose. Current findings show that our previous result of a 35% decrease in 3H/14C ratio in brain glucose 5 minutes after the injection was caused by undetected contaminants coeluted with glucose in the isolation process. Using an improved fractionation procedure in the present study we found that the rate of decrease of the 3H/14C ratio in brain glucose is significantly greater than that in blood glucose, the difference in slopes of the two time-courses was 29%. Quantitatively, the dephosphorylation of glucose-6-phosphate was studied from data obtained within the first minute after the injection of tracers. The formation of radioactive metabolites from [2-3H]-glucose is significantly greater than from [U-14C]-glucose immediately following the injection, 25% at 10 s, 21% at 20 s, and 14% at 30 s after the injection. The decreasing trend is caused by loss of tritiated water to blood circulation. The rate of glucose utilization calculated from the initial rates of formation of radioactive metabolites from [2-3H]-glucose was 1.04 umol/min/g; from [U-14C]-glucose was 0.83 umol/min/g. The difference of 0.21 mol/min/g was attributed to the dephosphorylation of glucose-6-phosphate in brain. Current findings reaffirm our previous conclusion that in brain, the dephosphorylation of glucose-6-phosphate is active. Because of rapid rate of glucose turnover in brain and the rapid equilibration of labeled glucose in the body aided by blood circulation, data obtained 2 min after the injection are not sufficient to conclude the quantitative aspect of the dephosphorylation of glucose-6-phosphate.